A T cell response is initiated by T cell receptor (TCR) activation through recognition of foreign antigen displayed on the surface of an antigen presenting cell (APC) and results in the directional secretion of certain cytokines or cytolytic factors towards the APC. Cytolytic factors cause death of an infected target cell, and cytokines stimulate other cells involved in the immune response. Directional secretion enables a T cell to specifically communicate with a target cell and is critical for effective T cell function. This is clearly demonstrated in diseases such as Griscelli syndrome and Chediak-Higashi syndrome where impaired directional secretion by T cells results in immunodeficiency. In addition, misdirected secretion could result in the death of uninfected cells or inappropriate activation of immune responses. Directional secretion requires reorientation of the T cell microtubule organizing center (MTOC) to the immune synapse (IS), the contact site with the target cell. Our understanding of MTOC reorientation has been limited by the difficulty of directiy observing the process. MTOC reorientation occurs within minutes of TCR activation, however most studies of this process are based on staining of fixed conjugates between T cells and APCs at set time points. This approach does not provide the temporal precision necessary for mechanistic study of such a rapid process. The goal of this project is to more clearly define the mechanism responsible for T cell MTOC reorientation using a photoactivatable antigen that is inactive until exposure to a short pulse of UV light. This method enables precise spatial and temporal control over T cell activation, and will be applied in single cell live imaging experiments to simultaneously observe recruitment behaviors of various fiuorescently labeled proteins and MTOC reorientation. It will also allow observation of how RNAi knockdown or chemical inhibition of a candidate protein affects MTOC reorientation and recruitment behaviors of other proteins in live T cells. Finally, a directional cytokine secretion assay will be used to determine how MTOC reorientation defects caused by knockdown or inhibition of proteins involved affects T cell function. PUBLIC HEATLH RELAVENCE: T cells play a central role in the antigen specific immune response. The ability to directionally secrete certain cytokines or cytolytic factors towards a target cell is critical for effective T cell function as it enables specific communication with a target cell. Directional secretion requires reorientation of the microtubule organizing center (MTOC) to the contact site with the target cell;however this process is poorly understood. I will investigate the mechanism of MTOC reorientation using a photo activatable antigen that provides precise control over T cell activation enables analysis with high spatial and temporal resolution.